- Email: [email protected]
Tracing Changes in the Microbial Community of a Hydrocarbon-Polluted Soil by Culture-Dependent Proteomics
Pedosphere 20(4): 479–485, 2010 ISSN 1002-0160/CN 32-1315/P c 2010 Soil Science Society of China Published by Elsevier Limited and Science Press
Tracing Changes in the Microbial Community of a Hydrocarbon-Polluted Soil by Culture-Dependent Proteomics∗1 ´ J. L. MORENO, T. HERNANDEZ ´ F. BASTIDA∗2 , C. NICOLAS, and C. GARC´IA Department of Soil and Water Conservation, Centro de Edafologia y Biologia Aplicada del Segura (CEBAS-CSIC), Campus Universitario de Espinardo, 30100 Espinardo, Murcia (Spain) (Received January 5, 2010; revised May 14, 2010)
ABSTRACT Hydrocarbon contamination may affect the soil microbial community, in terms of both diversity and function. A laboratory experiment was set-up, with a semi-arid control soil and the same soil but artificially contaminated with diesel oil, to follow changes in the dominant species of the microbial community in the hydrocarbon-polluted soil via proteomics. Analysis of the proteins extracted from enriched cultures growing in Luria-Bertani (LB) media showed a change in the microbial community. The majority of the proteins were related to glycolysis pathways, structural or protein synthesis. The results showed a relative increase in the complexity of the soil microbial community with hydrocarbon contamination, especially after 15 days of incubation. Species such as Ralstonia solanacearum, Synechococcus elongatus and different Clostridium sp. were adapted to contamination, not appearing in the control soil, although Bacillus sp. dominated the growing in LB in any of the treatments. We conclude that the identification of microbial species in soil extracts by culture-dependent proteomics is able to partially explain the changes in the diversity of the soil microbial community in hydrocarbon polluted semi-arid soils, but this information is much more limited than that provided by molecular methods. Key Words:
culture dependent, hydrocarbon contamination, microbial diversity, proteomics, semiarid soil
Citation: Bastida, F., Nicol´ as, C., Moreno, J. L., Hern´ andez, T. and Garc´ıa, C. 2010. Tracing changes in the microbial community of a hydrocarbon-polluted soil by culture-dependent proteomics. Pedosphere. 20(4): 479–485.
Hydrocarbon contamination may affect soil microbial structure. However, in soil microbiology there is a lack of knowledge regarding the environmental determinants of microbial population variation in soil environments contaminated by complex hydrocarbon mixtures (Hamamura et al., 2006). In this sense, different authors have observed, by denaturing gradient gel electrophoresis (DGGE) or terminalrestriction fragment length polymorphism (T-RFLP), a decrease in the microbial diversity of fuelcontaminated soils, leading to the predominance of well-adapted microorganisms (Ahn et al., 2006), and a change in the community structure (Labb´e et al., 2007). Recently, soil investigation has turned its efforts to the study of the real executors of the genome at molecular scales: the proteins (Wilmes and Bond, 2004), which provide direct evidence of the active microbial populations. Analysis of proteins from microbes by mass spectrometry and subsequent database processing can offer insights regarding both phylogeny and function of soil microorganisms. However, information related to soil proteomics for such contaminated soils is non-existent. Thus, our aim was to follow changes in the microbial community of a hydrocarbon-polluted, semi-arid soil via proteomics. MATERIALS AND METHODS Experimental design Samples were collected from the 0–20 cm layer of a soil in Santomera (38◦ 1 N, 1◦ 12 W), an ∗1 Supported
by the JAE-Program for Ph.D. Students of Spanish Research Council. author. E-mail: [email protected].
∗2 Corresponding
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experimental field area near to Murcia in southeastern Spain. This soil is characterised mainly by a cover of xerophytic shrubs such as Rhamnus lycoides and Thymus hyemalis L. The area from which the samples were taken has a semi-arid climate, with annual rainfall of 298 mm and a mean annual temperature of 17 ◦ C. The soil is a Calcic Kastanozems (Soil Survey Staff, 1998) and its main chemical characteristics are as follows (values on a dry weight basis): total organic carbon 33.7 g kg−1 ; electrical conductivity at 25 ◦ C 422 S cm−1 ; pH 7.38; total N (Kjeldhal) 3.50 g kg−1 ; total P 213 mg kg−1 ; total K 11.7 g kg−1 . Twelve sub-samples were taken from this soil and mixed to obtain the final sample. The experiment consisted of microcosm incubations under controlled temperature (25 ◦ C) and humidity. The treatment was an artificial contamination, achieved by mixing 500 g of soil with 25 mL of diesel fuel (source of hydrocarbons). A soil microcosm without fuel treatment was used as a control. The incubations were performed in triplicate for two incubation times: 7 h (T0) and 15 days (T15). All the treatments and the control were prepared in polyethylene containers and sufficient distilled water to reach 60% of soil water-holding capacity was added to each container. Periodically, the moisture content was controlled gravimetrically and distilled water added when necessary. The method described by Garc´ıa et al. (1997) was used to measure dehydrogenase activity, by using 1 g of soil, and the reduction of p-iodonitrotetrazolium chloride to p-iodonitrotetrazolium formazan. Microbial biomass C was determined by fumigation-extraction (Vance et al., 1987), using a Shimadzu TOC5050A total organic carbon analyzer. Protein separation and analysis Microbial inoculum from the soil treatments was obtained by mixing 10 g of soil with 100 mL 1:10 (w/v) phosphate buffered saline for 15 min. After centrifugation at 3 500 r min−1 for 10 min, 1 mL of supernatant was inoculated in liquid LB medium and cultured at 25 ◦ C at 200 r min−1 for 20 h. Longer time-frame incubation was not selected in order to restrict development of faster growing specie which may hamper the growing of the others. Microbial cells were centrifuged for 10 min at 8 000 r min−1 at 4 ◦ C. The supernatant was discarded and the pellet was washed twice with 50 mmol L−1 Tris-HCl buffer (pH 7.5) plus 0.1 mg mL−1 of chloranfenicol. 1.5 mL of the resuspended cells were centrifuged and re-suspended in 50 μL of sodium dodecyl sulphate (SDS) buffer, i.e., 20 mmol L−1 Tris-HCl of pH 7.5 with 0.2 g L−1 SDS. After a few seconds in a sonication bath, a heat shock was applied by shaking at 1 400 r min−1 at 60 ◦ C for 5 min. Cell pellets were treated with the protein inhibitor phenylmethylsulfonyl fluoride, lysed and processed as described elsewhere (Benndorf et al., 2007). Tryptic digestion was carried out directly on the protein extracts. Briefly, protein extracts were subjected to acetone precipitation. The pellet was re-suspended in 50 mmol L−1 ammonium bicarbonate buffer (pH 8.5) with 9% (v/v) acetonitrile and then digested with the following standard procedure. Samples were reduced with 10 mmol L−1 dithiothreitol (DTT) at 56 ◦ C for 15 min, and then were alkylated with 55 mmol L−1 iodoacetamide (IAA) for 1 h at room temperature in the dark. Excess IAA was quenched with 10 mmol L−1 DTT. Samples were then incubated with 0.4 μg of trypsin, proteomics grade (Sigma-Aldrich, reference T6567), for 16 h at 37 ◦ C. The reaction was stopped with 0.1% (v/v) formic acid and then samples were cleaned with C18 ZipTips (Millipore) and dried using a vacuum evaporator. For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 10 μL of solubilised proteins were mixed with sample buffer, incubated for 5 min at 60 ◦ C and loaded on SDS gels (4% stacking gel and 12% separating gel). After SDS-PAGE separation, proteins were visualised by colloidal Coomassie brilliant blue G-250 (CBB; Roth, Kassel, Germany) staining. Bands excised from gels were digested using trypsin, proteomics grade (Sigma-Aldrich, reference T6567), following the procedure of Izquierdo-Rico et al. (2009). High performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) analysis was used to identify the proteins both for protein extracts or SDS-PAGE bands. The analysis was carried out on an HPLC-MS system consisting of an Agilent 1100 Series HPLC (Agilent Tech-
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nologies, Santa Clara, CA) equipped with a μ-wellplate autosampler and a capillary pump, and an Agilent XCT Plus ion trap mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with an electrospray interface (ESI). The separation and analysis were performed as described elsewhere (Izquierdo-Rico et al., 2009). In order to cover the maximum species identification range, data processing was performed with the DataAnalysis programme for liquid chromatography (LC)-mass selective detector (MSD) trap version 3.2 (Bruker Daltonik, GmbH, Germany) and Spectrum Mill MS Proteomics Workbench (Agilent Technologies, Santa Clara, CA), and also using tandem mass spectrometry (MS/MS) ion search (Mascot, Matrix Science, London, UK) against all bacterial entries of NCBInr (GenBank) with the following parameters: trypsin digestion, up to two missed cleavage sites, fixed modifications of carbamidomethylation of cysteine, variable modifications of oxidation (methionine), peptide tolerance of 1.2 Da, MS/MS tolerance of 0.6 Da, and peptide charge of +1, +2 and +3. RESULTS AND DISCUSSION SDS-PAGE of proteins extracted directly from soil did not show the presence of any band in neither the control nor the polluted soil at two incubation times of T0 and T15 (data not shown). Until methods can be improved, the extraction of sufficient, clean proteins from soils will remain a handicap for protein identification and microbial species affiliation (Bastida et al., 2009). In this work, we suggest an alternative framework based on cell culture, as was used also by Rutz and Kieft (2004) for phylogenic characterization of semi-arid soils by molecular methods. Several authors have utilized enrichment cultures with different media, aiming to study functional processes and even diversity changes (Lehours et al., 2009). We hypothesize that culturable microbial species which are affected by gasoline contamination may be at a competitive disadvantage against other members of the microbial community which could be more resistant, and these changes can be studied by the extraction of proteins from liquid microbial cultures and their identification by mass spectrometry. It should be noted that our aim was not to study the vast soil microbial diversity (for this, molecular methods probably offer better results), but to know whether soil proteomics employing culture methods is able to identify both a shift in a soil microbial community and some species more-adapted to gasoline contamination, even when genome database information is scarce and may represent a handicap for microbial species affiliation of proteins from environmental samples (Keller and Hettich, 2009). For this, we should mention that our framework should cover a huge range for possible identification of many bacteria species in soil. We opted to make a search against two different searching engines (Mascot and Spectrum Mill from Agilent) with the aim of maximizing our results. In addition, two different electrophoresis techniques were carried out on the protein extracted from microbial cultures. The first is based on the analysis of crude protein extract by LC coupled to ESI-MS. This technique allows a separation of peptides after tryptic digestion and a consequent analysis in the ESI. The second technique is based on the excision of bands from SDS-PAGE gels followed by digestion and analysis by LC-ESIMS. In this case, only the major protein bands can be analysed. As a result of the experiment, about 19 bands were detected, both in the control and polluted soils at T0 and T15, with a nearly similar pattern of bands. However, a single SDS-PAGE band can contain more than one protein. This may be the reason for the similar pattern of the enzymatic pool of the microorganisms growing in LB (data not shown). For different microbial species, the enzyme pool growing in the same media could be nearly similar. Table I shows proteins arising from particular species of bacteria, both from SDS-PAGE bands and crude extract analysis. It can be said that the potentially-culturable soil bacterial community is dominated mainly by different Bacillus sp., such as B. anthracis, B. cereus or B. thuringiensis; these received the highest scores for the proteins after Mascot or Spectrum Mill analysis (Table I). Thus, scores up to 565 and coverage of nearly 30% were detected for formate acetyltransferase derived specifically from B. cereus. The majority of the proteins identified are related to cellular metabolism, such as
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TABLE I Protein identification and phylogeny information from the control and hydrocarbon contaminated soils (HC) at two incubation times of 7 h (T0) and 15 days (T15) Specific matching specie
Ascension Scorea) Sequence No. of peptides number coverage matched
Protein name
% Bacillus anthracis
Bacillus cereus
Bacillus halodurans Bacillus thuringiensis Symbiobacterium thermophilumb) B. anthracis
B. cereus
Bacillus subtilis B. thuringiensis Clostridium difficileb) Clostridium botulinumb) Bacillus weihenstephanensis B. anthracis
B. cereus
Clostridium perfringens Exiguobacterium sp.b) B. cereus
B. anthracis C. difficile B. thuringiensis C. perfringens B. subtilis B. weihenstephanensis Synechococcus elogatusb) a) Obtained
Control at T0 Phosphopyruvate hydratase 30265161 S-layer protein sap 30261020 Glyceraldehyde-3-phosphate dehydrogenase 30265165 Elongation factor Tu 30018378 Pyruvate dehydrogenase E1 component 30022060 beta subunit Formate acetyltransferase 42779643 Phosphopentomutase 15614093 Flagellin 189164121 Protein translation elongation factor G 51894213 Control Triosephosphate isomerase Phosphopyruvate hydratase Glyceraldehyde-3-phosphate dehydrogenase Formate acetyltransferase Elongation factor Tu 50S ribosomal protein L7/L12 30S ribosomal protein S11 Flagellin Hypothetical protein CdifQ 04003671 Formate acetyltransferase 1 Flagellin
464 217 91 169 117
28 26 29 10 42
6 16 6 3 8
118 80 154 62
18 3 38 1
10 1 11 2
+ HC at T0 65317109 242 30265161 203 30265164 166 47567276 565 30018378 272 30018370 61 16077210 69 189339436 473 145953667 70 148381164 68 87242498 95
19 44 46 26 16 29 22 24 7 2 21
3 12 11 13 4 4 1 5 1 1 6
18 50 42 9 6 9 36
5 14 8 4 1 1 7
4 2
1 1
7 28 15 33 24 4 4 42 10 12 4 37 5
2 8 7 7 7 2 1 7 2 2 1 14 1
Control at T15 Flagellin 30261766 309 Phosphopyruvate hydratase 30265161 225 Glyceraldehyde-3-phosphate dehydrogenase 30265165 122 Translation elongation factor G 218895242 263 Glyceraldehyde-3-phosphate dehydrogenase 30023174 110 30S ribosomal protein S7 30018376 85 Pyruvate dehydrogenase E1 component 30022060 82 beta subunit Elongation factor Tu 18311389 75 Hypothetical protein Exig 1335 172057364 57 Control + HC at T15 Elongation factor Tu 30018378 101 Phosphopyruvate hydratase 30023172 115 Formate acetyltransferase 30018697 197 Glyceraldehyde-3-phosphate dehydrogenase 30265165 113 Phosphopyruvate hydratase 30265161 134 Elongation factor Tu 126697624 148 Formate acetyltransferase 75760246 62 Flagellin 87242360 104 Hypothetical protein CPE0409 18309391 19 Pyruvate dehydrogenase (E1 beta subunit) 16078523 21 Glucose-6-phosphate isomerase 16080187 21 Phosphopyruvate hydratase 163942819 777 Sulfate transport system 56752413 55 substrate-binding protein
after searching against NCBInr;
b) Peptide
not found in replicate analysis.
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glycolysis pathways (phosphopyruvate hydratase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, etc.), or are structural proteins such as flagellin from B. thuringiensis. The presence of these proteins indicates a high microbial activity of these microorganisms. In addition, elongation factor G from Symbiobacterium thermophilum was also detected in the control soil at T0 (Table I). This protein is, in part, responsible for protein synthesis. S. thermophilum is a thermophilic bacterium found in a commensal submerged culture derived from compost (Suzuki et al., 1988). It is characterized by a marked growth dependence on microbial commensalism; it does not grow by itself under standard culture conditions. However, when co-cultured with Bacillus sp. strains, it propagates up to 5 × 108 cells mL−1 (Ohno et al., 1999), which supports our findings. Adequate conditions, which include an increased CO2 concentration and supply of peptides, could be created successfully by the simultaneous growth of cognate Bacillus sp. in laboratory culture (Ueda et al., 2004). The control treatment also showed a temporal variation, Clostridium perfringens and Exiguobacterium spp. being found after 15 days of incubation, apart from different Bacillus sp. Only one peptide was found to correspond specifically to Exiguobacterium sp., yet the probability based Mowse score from Mascot indicated identity or extensive homology at P < 0.05. In the case of C. Perfringens, the peptide sequence is “LLDEAQAGDNIGALLR” (1667.8842 mass [M+H]+ ), corresponding to elongation factor Tu (partly responsible for protein synthesis), which seems to be conserved in different Clostridium spp. A variation in the nutrient source or physico-chemical conditions could stimulate the development of different clostridia in the soil and hence in the liquid media. Different authors have observed a change in the structure and functionality of the microbial community after incubation under natural conditions (just irrigation and temperature) (Schimel et al., 1999; Pesaro et al., 2004). In fact, we observed a significant increase (P < 0.05) in the microbial biomass content after 15 days of incubation, compared with the control and contaminated soils at T0 (data not shown). Development of this and other species may be related to the observed increase in the size of the microbial community after incubation under adequate conditions (moisture and temperature), in part occupying the niche left by other less-adapted populations. Vivas et al. (2008) indicated that the complexity of a microbial community as well as the dehydrogenase activity negatively correlated with the soil contamination levels. Using culture dependent and independent methods, Phillips et al. (2006) proved that microbial community, influenced by plant specie, may affect hydrocarbon degradation in soil. Polluted soil showed a slight shift in the culturable microbial community. Proteins or peptides of up to five new species were found in the contaminated soil compared with the control at incubation times T0 and T15, respectively (Table I), even when some peptides could not be detected in replicates. It is important to note the identification of a peptide arising specifically from C. botulinum and C. perfringens at T0. This peptide originated from the digestion of an excised SDS-PAGE band. Clostridia, being motile bacteria that are ubiquitous in nature and especially prevalent in soil, have been identified by several authors using molecular methods (Weber et al., 2001). The development of these Gram-positive bacteria may be related to the soil resistance or resilience against negative factors, such as contamination, due to their sporulation capacity. After 15 days of incubation, Clostridium difficile, also appearing at T0, was presented in the contaminated soil. Identification of two different peptides specific to this microorganism, with a joint score of 148 (elongation factor Tu) (Table I), supports the prevalence of this species, suggesting its involvement in soil resistance against hydrocarbon contamination. However, the identification of proteins originating from C. perfringens may be related to the temporal changes of the natural soil and a response to irrigation, since this species appeared at day 15 in the control soil. Cyanobacteria develop as large, cryptic populations in the topsoil of arid land, where plant cover is restricted, water is scarce and harsh micro-environmental conditions prevail (Garc´ıa-Pinchel and Pringault, 2001). The identification of the cyanobacterium Synechococcus elongatus, sulphate transport system substrate-binding protein with the peptide sequence of “NFLFSFAKNIKTQVNSGR” (2069.8282 mass [M+H]+ ), may be related to water-drying pulses derived from irrigation for the maintenance of
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water-holding capacity. In any case, we only detected this peptide in one replicate and the presence of this and other specie should be verified by molecular methods. In addition, we detected the phosphopyruvate hydratase from Bacillus weihenstephanensis, which is involved in glycolysis pathways. Recently, Rajkumar et al. (2008) isolated a metal-resistant bacterium, characterised as B. weihenstephanensis by biochemical and 16S ribosomal DNA sequencing, from serpentine soils. The presence of this bacterium (and also some species of clostridia) in contaminated soils from the beginning (T0) can be explained by a change in the soil chemical conditions after contamination. Some dominant taxa may be diminished and their niche can be occupied by Clostridium spp. and B. weihenstephanensis, which probably are more adapted to these constrained conditions. This succession may be of paramount importance for the resilience and resistance of the soil microbial community against hydrocarbon contamination. In fact, these and other species not identified by our culture-dependent approach could be related to hydrocarbon degradation in contaminated soil or, at least, provide a reservoir of organic carbon able to be degraded by other species. Dehydrogenase activity was significantly higher (P < 0.05) in the contaminated soil (8.81 and 9.35 μg INTF g−1 soil h−1 for T0 and T15) than that in the control soil (7.99 and 7.75 μg INTF g−1 soil h−1 for T0 and T15). The significantly higher activity in the contaminated soil of this enzyme may be related to the degradation of hydrocarbon by the species prevalent after contamination. Other authors also have found the relations between hydrocarbon contamination and dehydrogenase activity (Dawson et al., 2007). In fact, the maintenance of an adequate microbial biomass in soil, with a high microbial activity, could be a mechanism for soil resistance to degradation factors and thus of paramount importance for ecosystem sustainability (Wertz et al., 2007; Banning and Murphy, 2008). Dehydrogenase activity has been defined as an enzymatic complex of intracellular nature related to the oxidation of organic compounds (Nannipieri et al., 1990). The existence of several dehydrogenases in soil, which are involved in intracellular processes of microorganisms, might be the origin of this enzyme. CONCLUSIONS Bacillus sp. dominated the microbial community both in the control and hydrocarbon polluted soils. However, for the first time, the mere identification of microbial proteins by culture-dependent proteomics of soil extracts could suggest some minor changes in the soil microbial community diversity that could be related to phenomena of resistance and resilience against short-term hydrocarbon contamination in semi-arid areas. The presence of single peptides has to be taken carefully as a proof of the presence of the host microorganisms. However, much more effort should be made to address protein extraction from bulk soil. Application of proteomics to soil science is an open door to the study of the functional role of microbial species, although probably changes in microbial diversity are better achieved by molecular methods. ACKNOWLEDGEMENTS The authors thank Dr. A. TORRECILLAS from University of Murcia, Spain for his technical and ´ thanks the Spanish Research Council (CSIC) for his Ph.D. stutheoretical support. Dr. C. NICOLAS dentship. REFERENCES Ahn, J. H., Kim, M. S., Kim, M. C., Lim, J. S., Lee, G. T., Yun, J. K., Kim, T., Kim, T. and Ka, J. O. 2006. Analysis of bacterial diversity and community structure in forest soils contaminated with fuel hydrocarbon. J. Microbiol. Biotechn. 16: 704–715. Banning, N. C. and Murphy, D. V. 2008. Effect of heat-induced disturbance on microbial biomass and activity in forest soil and the relationship between disturbance effects and microbial community structure. Appl. Soil Ecol. 40: 109–119. Bastida, F., Moreno, J. L., Nicol´ as, C., Hern´ andez, T. and Garc´ıa, C. 2009. Soil metaproteomics: a review of an emerging environmental science. Significance, methodology and perspectives. Eur. J. Soil Sci. 60: 845–859.
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Benndorf, D., Balcke, G. U., Harms, H. and von Bergen, M. 2007. Functional metaproteome analysis of protein extracts from contaminated soil and groundwater. The ISME J. 1: 224–234. Dawson, J. J. C., Godsiffe, E. J., Thompson, I. P., Ralebitso-Senior, T. K., Killham, K. S. and Paton, G. I. 2007. Application of biological indicators to assess recovery of hydrocarbon impacted soils. Soil Biol. Biochem. 39: 164–177. Garc´ıa, C., Rold´ an, A. and Hern´ andez, T. 1997. Changes in microbial activity after abandonment of cultivation in a semiarid Mediterranean environment. J. Environ. Qual. 26: 285–291. Garc´ıa-Pichel, F. and Pringault, O. 2001. Microbiology: Cyanobacteria track water in desert soils. Nature. 413: 380–381. Hamamura, N., Olson, S. H., Ward, D. M. and Inskeep, W. P. 2006. Microbial population dynamics associated with crude-oil biodegradation in diverse soils. Appl. Environ. Microb. 72: 6316–6324. Izquierdo-Rico, M. J., Jim´enez-Movilla, M., Llop, E., P´ erez-Oliva, A. B., Ballesta, J., Gut´ıerrez-Gallego, R., Jim´enezCervantes, C. and Avil´es, M. 2009. Hamster zona pellucida is formed by four glycoproteins: ZP1, ZP2, ZP3, and ZP4. J. Proteome. Res. 8: 926–941. Keller, M. and Hettich, R. 2009. Environmental proteomics: a paradigm shift in characterizing microbial activities at the molecular level. Microbiol. Mol. Biol. R. 73: 62–70. Labb` e, D., Margesin, R., Schinner, F., Whitten, L. G. and Greer, C. W. 2007. Comparative phylogenetic analysis of microbial communities in pristine and hydrocarbon-contaminated Alpine soils. FEMS Microbiol. Ecol. 59: 466–475. Lehours, A.-C., Batisson, I., Guedon, A., Mailhot, G. and Fonty, G. 2009. Diversity of culturable bacteria, from the anaerobic zone of the meromictic Lake Pavin, able to perform dissimilatory-iron reduction in different in vitro conditions. Geomicrobiol. J. 26: 212–223. Nannipieri, P., Grego, S. and Ceccanti, B. 1990. Ecological significance of the biological activity in soils. In Bollag, J. M. and Stotzky, G. (eds.) Soil Biochemistry. Marcel Dekker, New York. pp. 293–355. Ohno, M., Okano, I., Watsuji, T., Kakinuma, T., Ueda, K. and Beppu, T. 1999. Establishing the independent culture of a strictly symbiotic bacterium Symbiobacterium thermophilum from its supporting Bacillus strain. Biosci. Biotech. Bioch. 63: 1083–1090. Pesaro, M., Nicollier, G., Zeyer, J. and Widmer, F. 2004. Impact of soil drying-rewetting stress on microbial communities and activities and on degradation of two crop protection products. Appl. Environ. Microb. 70: 2577–2587. Phillips, L. A., Greer, C. W. and Germida, J. J. 2006. Culture-based and culture-independent assessment of the impact of mixed and single plant treatments on rhizosphere microbial communities in hydrocarbon contaminated flare-pit soil. Soil Biol. Biochem. 38: 2823–2833. Rajkumar, M., Ma, Y. and Freitas, H. 2008. Characterization of metal-resistant plant-growth promoting Bacillus weihenstephanensis isolated from serpentine soil in Portugal. J. Basic Microb. 48: 500–508. Rutz, B. A. and Kieft, T. L. 2004. Phylogenetic characterization of dwarf archaea and bacteria from a semiarid soil. Soil Biol. Biochem. 36: 825–833. Schimel, J. P., Gulledge, J. M., Clein-Curley, J. S., Lindstrom, J. E. and Braddock, J. F. 1999. Moisture effects on microbial activity and community structure in decomposing birch litter in the Alaskan taiga. Soil Biol. Biochem. 31: 831–838. Soil Survey Staff. 1998. Keys of Soil Taxonomy. 8th Edition. USDA-NRCS, Washington, DC. Suzuki, S., Horinouchi, S. and Beppu, T. 1988. Growth of a tryptophanase-producing thermophile, Symbiobacterium thermophilum gen. nov., sp. nov., is dependent on co-culture with a Bacillus sp. J. Gen. Microbiol. 134: 2353–2362. Ueda, K., Yamashita, A., Ishikawa, J., Shimada, M., Watsuji, T. O., Morimura, K., Ikeda, H., Hattori, M. and Beppu, T. 2004. Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism. Nucleic Acids Res. 32: 4937–4944. Vance, E. D., Brookes, P. C. and Jenkinson, D. S. 1987. An extraction method for measuring soil microbial biomass C. Soil Biol. Biochem. 19: 703–707. Vivas, A., Moreno, B., del Val, C., Macci, C., Masciandaro, G. and Ben´ıtez, E. 2008. Metabolic and bacterial diversity in soils historically contaminated by heavy metals and hydrocarbons. J. Environ. Monitor. 10: 1287–1296. Weber, S., Stubner, S. and Conrad, R. 2001. Bacterial populations colonizing and degrading rice straw in anoxic paddy soil. Appl. Environ. Microb. 67: 1318–1327. Wertz, S., Degrange, V., Prosser, J. I., Poly, F., Commeaux, C., Guillaumaud, N. and Le Roux, X. 2007. Decline of soil microbial diversity does not influence the resistance and resilience of key soil microbial functional groups following a model disturbance. Environ. Microbiol. 9: 2211–2219. Wilmes, P. and Bond, P. L. 2004. The application of two-dimensional polyacrylamide gel electrophoresis and downstream analyses to a mixed community of prokaryotic microorganisms. Environ. Microbiol. 6: 911–920.
Tracing Changes in the Microbial Community of a Hydrocarbon-Polluted Soil by Culture-Dependent Proteomics∗1 ´ J. L. MORENO, T. HERNANDEZ ´ F. BASTIDA∗2 , C. NICOLAS, and C. GARC´IA Department of Soil and Water Conservation, Centro de Edafologia y Biologia Aplicada del Segura (CEBAS-CSIC), Campus Universitario de Espinardo, 30100 Espinardo, Murcia (Spain) (Received January 5, 2010; revised May 14, 2010)
ABSTRACT Hydrocarbon contamination may affect the soil microbial community, in terms of both diversity and function. A laboratory experiment was set-up, with a semi-arid control soil and the same soil but artificially contaminated with diesel oil, to follow changes in the dominant species of the microbial community in the hydrocarbon-polluted soil via proteomics. Analysis of the proteins extracted from enriched cultures growing in Luria-Bertani (LB) media showed a change in the microbial community. The majority of the proteins were related to glycolysis pathways, structural or protein synthesis. The results showed a relative increase in the complexity of the soil microbial community with hydrocarbon contamination, especially after 15 days of incubation. Species such as Ralstonia solanacearum, Synechococcus elongatus and different Clostridium sp. were adapted to contamination, not appearing in the control soil, although Bacillus sp. dominated the growing in LB in any of the treatments. We conclude that the identification of microbial species in soil extracts by culture-dependent proteomics is able to partially explain the changes in the diversity of the soil microbial community in hydrocarbon polluted semi-arid soils, but this information is much more limited than that provided by molecular methods. Key Words:
culture dependent, hydrocarbon contamination, microbial diversity, proteomics, semiarid soil
Citation: Bastida, F., Nicol´ as, C., Moreno, J. L., Hern´ andez, T. and Garc´ıa, C. 2010. Tracing changes in the microbial community of a hydrocarbon-polluted soil by culture-dependent proteomics. Pedosphere. 20(4): 479–485.
Hydrocarbon contamination may affect soil microbial structure. However, in soil microbiology there is a lack of knowledge regarding the environmental determinants of microbial population variation in soil environments contaminated by complex hydrocarbon mixtures (Hamamura et al., 2006). In this sense, different authors have observed, by denaturing gradient gel electrophoresis (DGGE) or terminalrestriction fragment length polymorphism (T-RFLP), a decrease in the microbial diversity of fuelcontaminated soils, leading to the predominance of well-adapted microorganisms (Ahn et al., 2006), and a change in the community structure (Labb´e et al., 2007). Recently, soil investigation has turned its efforts to the study of the real executors of the genome at molecular scales: the proteins (Wilmes and Bond, 2004), which provide direct evidence of the active microbial populations. Analysis of proteins from microbes by mass spectrometry and subsequent database processing can offer insights regarding both phylogeny and function of soil microorganisms. However, information related to soil proteomics for such contaminated soils is non-existent. Thus, our aim was to follow changes in the microbial community of a hydrocarbon-polluted, semi-arid soil via proteomics. MATERIALS AND METHODS Experimental design Samples were collected from the 0–20 cm layer of a soil in Santomera (38◦ 1 N, 1◦ 12 W), an ∗1 Supported
by the JAE-Program for Ph.D. Students of Spanish Research Council. author. E-mail: [email protected].
∗2 Corresponding
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experimental field area near to Murcia in southeastern Spain. This soil is characterised mainly by a cover of xerophytic shrubs such as Rhamnus lycoides and Thymus hyemalis L. The area from which the samples were taken has a semi-arid climate, with annual rainfall of 298 mm and a mean annual temperature of 17 ◦ C. The soil is a Calcic Kastanozems (Soil Survey Staff, 1998) and its main chemical characteristics are as follows (values on a dry weight basis): total organic carbon 33.7 g kg−1 ; electrical conductivity at 25 ◦ C 422 S cm−1 ; pH 7.38; total N (Kjeldhal) 3.50 g kg−1 ; total P 213 mg kg−1 ; total K 11.7 g kg−1 . Twelve sub-samples were taken from this soil and mixed to obtain the final sample. The experiment consisted of microcosm incubations under controlled temperature (25 ◦ C) and humidity. The treatment was an artificial contamination, achieved by mixing 500 g of soil with 25 mL of diesel fuel (source of hydrocarbons). A soil microcosm without fuel treatment was used as a control. The incubations were performed in triplicate for two incubation times: 7 h (T0) and 15 days (T15). All the treatments and the control were prepared in polyethylene containers and sufficient distilled water to reach 60% of soil water-holding capacity was added to each container. Periodically, the moisture content was controlled gravimetrically and distilled water added when necessary. The method described by Garc´ıa et al. (1997) was used to measure dehydrogenase activity, by using 1 g of soil, and the reduction of p-iodonitrotetrazolium chloride to p-iodonitrotetrazolium formazan. Microbial biomass C was determined by fumigation-extraction (Vance et al., 1987), using a Shimadzu TOC5050A total organic carbon analyzer. Protein separation and analysis Microbial inoculum from the soil treatments was obtained by mixing 10 g of soil with 100 mL 1:10 (w/v) phosphate buffered saline for 15 min. After centrifugation at 3 500 r min−1 for 10 min, 1 mL of supernatant was inoculated in liquid LB medium and cultured at 25 ◦ C at 200 r min−1 for 20 h. Longer time-frame incubation was not selected in order to restrict development of faster growing specie which may hamper the growing of the others. Microbial cells were centrifuged for 10 min at 8 000 r min−1 at 4 ◦ C. The supernatant was discarded and the pellet was washed twice with 50 mmol L−1 Tris-HCl buffer (pH 7.5) plus 0.1 mg mL−1 of chloranfenicol. 1.5 mL of the resuspended cells were centrifuged and re-suspended in 50 μL of sodium dodecyl sulphate (SDS) buffer, i.e., 20 mmol L−1 Tris-HCl of pH 7.5 with 0.2 g L−1 SDS. After a few seconds in a sonication bath, a heat shock was applied by shaking at 1 400 r min−1 at 60 ◦ C for 5 min. Cell pellets were treated with the protein inhibitor phenylmethylsulfonyl fluoride, lysed and processed as described elsewhere (Benndorf et al., 2007). Tryptic digestion was carried out directly on the protein extracts. Briefly, protein extracts were subjected to acetone precipitation. The pellet was re-suspended in 50 mmol L−1 ammonium bicarbonate buffer (pH 8.5) with 9% (v/v) acetonitrile and then digested with the following standard procedure. Samples were reduced with 10 mmol L−1 dithiothreitol (DTT) at 56 ◦ C for 15 min, and then were alkylated with 55 mmol L−1 iodoacetamide (IAA) for 1 h at room temperature in the dark. Excess IAA was quenched with 10 mmol L−1 DTT. Samples were then incubated with 0.4 μg of trypsin, proteomics grade (Sigma-Aldrich, reference T6567), for 16 h at 37 ◦ C. The reaction was stopped with 0.1% (v/v) formic acid and then samples were cleaned with C18 ZipTips (Millipore) and dried using a vacuum evaporator. For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 10 μL of solubilised proteins were mixed with sample buffer, incubated for 5 min at 60 ◦ C and loaded on SDS gels (4% stacking gel and 12% separating gel). After SDS-PAGE separation, proteins were visualised by colloidal Coomassie brilliant blue G-250 (CBB; Roth, Kassel, Germany) staining. Bands excised from gels were digested using trypsin, proteomics grade (Sigma-Aldrich, reference T6567), following the procedure of Izquierdo-Rico et al. (2009). High performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) analysis was used to identify the proteins both for protein extracts or SDS-PAGE bands. The analysis was carried out on an HPLC-MS system consisting of an Agilent 1100 Series HPLC (Agilent Tech-
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nologies, Santa Clara, CA) equipped with a μ-wellplate autosampler and a capillary pump, and an Agilent XCT Plus ion trap mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with an electrospray interface (ESI). The separation and analysis were performed as described elsewhere (Izquierdo-Rico et al., 2009). In order to cover the maximum species identification range, data processing was performed with the DataAnalysis programme for liquid chromatography (LC)-mass selective detector (MSD) trap version 3.2 (Bruker Daltonik, GmbH, Germany) and Spectrum Mill MS Proteomics Workbench (Agilent Technologies, Santa Clara, CA), and also using tandem mass spectrometry (MS/MS) ion search (Mascot, Matrix Science, London, UK) against all bacterial entries of NCBInr (GenBank) with the following parameters: trypsin digestion, up to two missed cleavage sites, fixed modifications of carbamidomethylation of cysteine, variable modifications of oxidation (methionine), peptide tolerance of 1.2 Da, MS/MS tolerance of 0.6 Da, and peptide charge of +1, +2 and +3. RESULTS AND DISCUSSION SDS-PAGE of proteins extracted directly from soil did not show the presence of any band in neither the control nor the polluted soil at two incubation times of T0 and T15 (data not shown). Until methods can be improved, the extraction of sufficient, clean proteins from soils will remain a handicap for protein identification and microbial species affiliation (Bastida et al., 2009). In this work, we suggest an alternative framework based on cell culture, as was used also by Rutz and Kieft (2004) for phylogenic characterization of semi-arid soils by molecular methods. Several authors have utilized enrichment cultures with different media, aiming to study functional processes and even diversity changes (Lehours et al., 2009). We hypothesize that culturable microbial species which are affected by gasoline contamination may be at a competitive disadvantage against other members of the microbial community which could be more resistant, and these changes can be studied by the extraction of proteins from liquid microbial cultures and their identification by mass spectrometry. It should be noted that our aim was not to study the vast soil microbial diversity (for this, molecular methods probably offer better results), but to know whether soil proteomics employing culture methods is able to identify both a shift in a soil microbial community and some species more-adapted to gasoline contamination, even when genome database information is scarce and may represent a handicap for microbial species affiliation of proteins from environmental samples (Keller and Hettich, 2009). For this, we should mention that our framework should cover a huge range for possible identification of many bacteria species in soil. We opted to make a search against two different searching engines (Mascot and Spectrum Mill from Agilent) with the aim of maximizing our results. In addition, two different electrophoresis techniques were carried out on the protein extracted from microbial cultures. The first is based on the analysis of crude protein extract by LC coupled to ESI-MS. This technique allows a separation of peptides after tryptic digestion and a consequent analysis in the ESI. The second technique is based on the excision of bands from SDS-PAGE gels followed by digestion and analysis by LC-ESIMS. In this case, only the major protein bands can be analysed. As a result of the experiment, about 19 bands were detected, both in the control and polluted soils at T0 and T15, with a nearly similar pattern of bands. However, a single SDS-PAGE band can contain more than one protein. This may be the reason for the similar pattern of the enzymatic pool of the microorganisms growing in LB (data not shown). For different microbial species, the enzyme pool growing in the same media could be nearly similar. Table I shows proteins arising from particular species of bacteria, both from SDS-PAGE bands and crude extract analysis. It can be said that the potentially-culturable soil bacterial community is dominated mainly by different Bacillus sp., such as B. anthracis, B. cereus or B. thuringiensis; these received the highest scores for the proteins after Mascot or Spectrum Mill analysis (Table I). Thus, scores up to 565 and coverage of nearly 30% were detected for formate acetyltransferase derived specifically from B. cereus. The majority of the proteins identified are related to cellular metabolism, such as
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TABLE I Protein identification and phylogeny information from the control and hydrocarbon contaminated soils (HC) at two incubation times of 7 h (T0) and 15 days (T15) Specific matching specie
Ascension Scorea) Sequence No. of peptides number coverage matched
Protein name
% Bacillus anthracis
Bacillus cereus
Bacillus halodurans Bacillus thuringiensis Symbiobacterium thermophilumb) B. anthracis
B. cereus
Bacillus subtilis B. thuringiensis Clostridium difficileb) Clostridium botulinumb) Bacillus weihenstephanensis B. anthracis
B. cereus
Clostridium perfringens Exiguobacterium sp.b) B. cereus
B. anthracis C. difficile B. thuringiensis C. perfringens B. subtilis B. weihenstephanensis Synechococcus elogatusb) a) Obtained
Control at T0 Phosphopyruvate hydratase 30265161 S-layer protein sap 30261020 Glyceraldehyde-3-phosphate dehydrogenase 30265165 Elongation factor Tu 30018378 Pyruvate dehydrogenase E1 component 30022060 beta subunit Formate acetyltransferase 42779643 Phosphopentomutase 15614093 Flagellin 189164121 Protein translation elongation factor G 51894213 Control Triosephosphate isomerase Phosphopyruvate hydratase Glyceraldehyde-3-phosphate dehydrogenase Formate acetyltransferase Elongation factor Tu 50S ribosomal protein L7/L12 30S ribosomal protein S11 Flagellin Hypothetical protein CdifQ 04003671 Formate acetyltransferase 1 Flagellin
464 217 91 169 117
28 26 29 10 42
6 16 6 3 8
118 80 154 62
18 3 38 1
10 1 11 2
+ HC at T0 65317109 242 30265161 203 30265164 166 47567276 565 30018378 272 30018370 61 16077210 69 189339436 473 145953667 70 148381164 68 87242498 95
19 44 46 26 16 29 22 24 7 2 21
3 12 11 13 4 4 1 5 1 1 6
18 50 42 9 6 9 36
5 14 8 4 1 1 7
4 2
1 1
7 28 15 33 24 4 4 42 10 12 4 37 5
2 8 7 7 7 2 1 7 2 2 1 14 1
Control at T15 Flagellin 30261766 309 Phosphopyruvate hydratase 30265161 225 Glyceraldehyde-3-phosphate dehydrogenase 30265165 122 Translation elongation factor G 218895242 263 Glyceraldehyde-3-phosphate dehydrogenase 30023174 110 30S ribosomal protein S7 30018376 85 Pyruvate dehydrogenase E1 component 30022060 82 beta subunit Elongation factor Tu 18311389 75 Hypothetical protein Exig 1335 172057364 57 Control + HC at T15 Elongation factor Tu 30018378 101 Phosphopyruvate hydratase 30023172 115 Formate acetyltransferase 30018697 197 Glyceraldehyde-3-phosphate dehydrogenase 30265165 113 Phosphopyruvate hydratase 30265161 134 Elongation factor Tu 126697624 148 Formate acetyltransferase 75760246 62 Flagellin 87242360 104 Hypothetical protein CPE0409 18309391 19 Pyruvate dehydrogenase (E1 beta subunit) 16078523 21 Glucose-6-phosphate isomerase 16080187 21 Phosphopyruvate hydratase 163942819 777 Sulfate transport system 56752413 55 substrate-binding protein
after searching against NCBInr;
b) Peptide
not found in replicate analysis.
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glycolysis pathways (phosphopyruvate hydratase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, etc.), or are structural proteins such as flagellin from B. thuringiensis. The presence of these proteins indicates a high microbial activity of these microorganisms. In addition, elongation factor G from Symbiobacterium thermophilum was also detected in the control soil at T0 (Table I). This protein is, in part, responsible for protein synthesis. S. thermophilum is a thermophilic bacterium found in a commensal submerged culture derived from compost (Suzuki et al., 1988). It is characterized by a marked growth dependence on microbial commensalism; it does not grow by itself under standard culture conditions. However, when co-cultured with Bacillus sp. strains, it propagates up to 5 × 108 cells mL−1 (Ohno et al., 1999), which supports our findings. Adequate conditions, which include an increased CO2 concentration and supply of peptides, could be created successfully by the simultaneous growth of cognate Bacillus sp. in laboratory culture (Ueda et al., 2004). The control treatment also showed a temporal variation, Clostridium perfringens and Exiguobacterium spp. being found after 15 days of incubation, apart from different Bacillus sp. Only one peptide was found to correspond specifically to Exiguobacterium sp., yet the probability based Mowse score from Mascot indicated identity or extensive homology at P < 0.05. In the case of C. Perfringens, the peptide sequence is “LLDEAQAGDNIGALLR” (1667.8842 mass [M+H]+ ), corresponding to elongation factor Tu (partly responsible for protein synthesis), which seems to be conserved in different Clostridium spp. A variation in the nutrient source or physico-chemical conditions could stimulate the development of different clostridia in the soil and hence in the liquid media. Different authors have observed a change in the structure and functionality of the microbial community after incubation under natural conditions (just irrigation and temperature) (Schimel et al., 1999; Pesaro et al., 2004). In fact, we observed a significant increase (P < 0.05) in the microbial biomass content after 15 days of incubation, compared with the control and contaminated soils at T0 (data not shown). Development of this and other species may be related to the observed increase in the size of the microbial community after incubation under adequate conditions (moisture and temperature), in part occupying the niche left by other less-adapted populations. Vivas et al. (2008) indicated that the complexity of a microbial community as well as the dehydrogenase activity negatively correlated with the soil contamination levels. Using culture dependent and independent methods, Phillips et al. (2006) proved that microbial community, influenced by plant specie, may affect hydrocarbon degradation in soil. Polluted soil showed a slight shift in the culturable microbial community. Proteins or peptides of up to five new species were found in the contaminated soil compared with the control at incubation times T0 and T15, respectively (Table I), even when some peptides could not be detected in replicates. It is important to note the identification of a peptide arising specifically from C. botulinum and C. perfringens at T0. This peptide originated from the digestion of an excised SDS-PAGE band. Clostridia, being motile bacteria that are ubiquitous in nature and especially prevalent in soil, have been identified by several authors using molecular methods (Weber et al., 2001). The development of these Gram-positive bacteria may be related to the soil resistance or resilience against negative factors, such as contamination, due to their sporulation capacity. After 15 days of incubation, Clostridium difficile, also appearing at T0, was presented in the contaminated soil. Identification of two different peptides specific to this microorganism, with a joint score of 148 (elongation factor Tu) (Table I), supports the prevalence of this species, suggesting its involvement in soil resistance against hydrocarbon contamination. However, the identification of proteins originating from C. perfringens may be related to the temporal changes of the natural soil and a response to irrigation, since this species appeared at day 15 in the control soil. Cyanobacteria develop as large, cryptic populations in the topsoil of arid land, where plant cover is restricted, water is scarce and harsh micro-environmental conditions prevail (Garc´ıa-Pinchel and Pringault, 2001). The identification of the cyanobacterium Synechococcus elongatus, sulphate transport system substrate-binding protein with the peptide sequence of “NFLFSFAKNIKTQVNSGR” (2069.8282 mass [M+H]+ ), may be related to water-drying pulses derived from irrigation for the maintenance of
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water-holding capacity. In any case, we only detected this peptide in one replicate and the presence of this and other specie should be verified by molecular methods. In addition, we detected the phosphopyruvate hydratase from Bacillus weihenstephanensis, which is involved in glycolysis pathways. Recently, Rajkumar et al. (2008) isolated a metal-resistant bacterium, characterised as B. weihenstephanensis by biochemical and 16S ribosomal DNA sequencing, from serpentine soils. The presence of this bacterium (and also some species of clostridia) in contaminated soils from the beginning (T0) can be explained by a change in the soil chemical conditions after contamination. Some dominant taxa may be diminished and their niche can be occupied by Clostridium spp. and B. weihenstephanensis, which probably are more adapted to these constrained conditions. This succession may be of paramount importance for the resilience and resistance of the soil microbial community against hydrocarbon contamination. In fact, these and other species not identified by our culture-dependent approach could be related to hydrocarbon degradation in contaminated soil or, at least, provide a reservoir of organic carbon able to be degraded by other species. Dehydrogenase activity was significantly higher (P < 0.05) in the contaminated soil (8.81 and 9.35 μg INTF g−1 soil h−1 for T0 and T15) than that in the control soil (7.99 and 7.75 μg INTF g−1 soil h−1 for T0 and T15). The significantly higher activity in the contaminated soil of this enzyme may be related to the degradation of hydrocarbon by the species prevalent after contamination. Other authors also have found the relations between hydrocarbon contamination and dehydrogenase activity (Dawson et al., 2007). In fact, the maintenance of an adequate microbial biomass in soil, with a high microbial activity, could be a mechanism for soil resistance to degradation factors and thus of paramount importance for ecosystem sustainability (Wertz et al., 2007; Banning and Murphy, 2008). Dehydrogenase activity has been defined as an enzymatic complex of intracellular nature related to the oxidation of organic compounds (Nannipieri et al., 1990). The existence of several dehydrogenases in soil, which are involved in intracellular processes of microorganisms, might be the origin of this enzyme. CONCLUSIONS Bacillus sp. dominated the microbial community both in the control and hydrocarbon polluted soils. However, for the first time, the mere identification of microbial proteins by culture-dependent proteomics of soil extracts could suggest some minor changes in the soil microbial community diversity that could be related to phenomena of resistance and resilience against short-term hydrocarbon contamination in semi-arid areas. The presence of single peptides has to be taken carefully as a proof of the presence of the host microorganisms. However, much more effort should be made to address protein extraction from bulk soil. Application of proteomics to soil science is an open door to the study of the functional role of microbial species, although probably changes in microbial diversity are better achieved by molecular methods. ACKNOWLEDGEMENTS The authors thank Dr. A. TORRECILLAS from University of Murcia, Spain for his technical and ´ thanks the Spanish Research Council (CSIC) for his Ph.D. stutheoretical support. Dr. C. NICOLAS dentship. REFERENCES Ahn, J. H., Kim, M. S., Kim, M. C., Lim, J. S., Lee, G. T., Yun, J. K., Kim, T., Kim, T. and Ka, J. O. 2006. Analysis of bacterial diversity and community structure in forest soils contaminated with fuel hydrocarbon. J. Microbiol. Biotechn. 16: 704–715. Banning, N. C. and Murphy, D. V. 2008. Effect of heat-induced disturbance on microbial biomass and activity in forest soil and the relationship between disturbance effects and microbial community structure. Appl. Soil Ecol. 40: 109–119. Bastida, F., Moreno, J. L., Nicol´ as, C., Hern´ andez, T. and Garc´ıa, C. 2009. Soil metaproteomics: a review of an emerging environmental science. Significance, methodology and perspectives. Eur. J. Soil Sci. 60: 845–859.
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Benndorf, D., Balcke, G. U., Harms, H. and von Bergen, M. 2007. Functional metaproteome analysis of protein extracts from contaminated soil and groundwater. The ISME J. 1: 224–234. Dawson, J. J. C., Godsiffe, E. J., Thompson, I. P., Ralebitso-Senior, T. K., Killham, K. S. and Paton, G. I. 2007. Application of biological indicators to assess recovery of hydrocarbon impacted soils. Soil Biol. Biochem. 39: 164–177. Garc´ıa, C., Rold´ an, A. and Hern´ andez, T. 1997. Changes in microbial activity after abandonment of cultivation in a semiarid Mediterranean environment. J. Environ. Qual. 26: 285–291. Garc´ıa-Pichel, F. and Pringault, O. 2001. Microbiology: Cyanobacteria track water in desert soils. Nature. 413: 380–381. Hamamura, N., Olson, S. H., Ward, D. M. and Inskeep, W. P. 2006. Microbial population dynamics associated with crude-oil biodegradation in diverse soils. Appl. Environ. Microb. 72: 6316–6324. Izquierdo-Rico, M. J., Jim´enez-Movilla, M., Llop, E., P´ erez-Oliva, A. B., Ballesta, J., Gut´ıerrez-Gallego, R., Jim´enezCervantes, C. and Avil´es, M. 2009. Hamster zona pellucida is formed by four glycoproteins: ZP1, ZP2, ZP3, and ZP4. J. Proteome. Res. 8: 926–941. Keller, M. and Hettich, R. 2009. Environmental proteomics: a paradigm shift in characterizing microbial activities at the molecular level. Microbiol. Mol. Biol. R. 73: 62–70. Labb` e, D., Margesin, R., Schinner, F., Whitten, L. G. and Greer, C. W. 2007. Comparative phylogenetic analysis of microbial communities in pristine and hydrocarbon-contaminated Alpine soils. FEMS Microbiol. Ecol. 59: 466–475. Lehours, A.-C., Batisson, I., Guedon, A., Mailhot, G. and Fonty, G. 2009. Diversity of culturable bacteria, from the anaerobic zone of the meromictic Lake Pavin, able to perform dissimilatory-iron reduction in different in vitro conditions. Geomicrobiol. J. 26: 212–223. Nannipieri, P., Grego, S. and Ceccanti, B. 1990. Ecological significance of the biological activity in soils. In Bollag, J. M. and Stotzky, G. (eds.) Soil Biochemistry. Marcel Dekker, New York. pp. 293–355. Ohno, M., Okano, I., Watsuji, T., Kakinuma, T., Ueda, K. and Beppu, T. 1999. Establishing the independent culture of a strictly symbiotic bacterium Symbiobacterium thermophilum from its supporting Bacillus strain. Biosci. Biotech. Bioch. 63: 1083–1090. Pesaro, M., Nicollier, G., Zeyer, J. and Widmer, F. 2004. Impact of soil drying-rewetting stress on microbial communities and activities and on degradation of two crop protection products. Appl. Environ. Microb. 70: 2577–2587. Phillips, L. A., Greer, C. W. and Germida, J. J. 2006. Culture-based and culture-independent assessment of the impact of mixed and single plant treatments on rhizosphere microbial communities in hydrocarbon contaminated flare-pit soil. Soil Biol. Biochem. 38: 2823–2833. Rajkumar, M., Ma, Y. and Freitas, H. 2008. Characterization of metal-resistant plant-growth promoting Bacillus weihenstephanensis isolated from serpentine soil in Portugal. J. Basic Microb. 48: 500–508. Rutz, B. A. and Kieft, T. L. 2004. Phylogenetic characterization of dwarf archaea and bacteria from a semiarid soil. Soil Biol. Biochem. 36: 825–833. Schimel, J. P., Gulledge, J. M., Clein-Curley, J. S., Lindstrom, J. E. and Braddock, J. F. 1999. Moisture effects on microbial activity and community structure in decomposing birch litter in the Alaskan taiga. Soil Biol. Biochem. 31: 831–838. Soil Survey Staff. 1998. Keys of Soil Taxonomy. 8th Edition. USDA-NRCS, Washington, DC. Suzuki, S., Horinouchi, S. and Beppu, T. 1988. Growth of a tryptophanase-producing thermophile, Symbiobacterium thermophilum gen. nov., sp. nov., is dependent on co-culture with a Bacillus sp. J. Gen. Microbiol. 134: 2353–2362. Ueda, K., Yamashita, A., Ishikawa, J., Shimada, M., Watsuji, T. O., Morimura, K., Ikeda, H., Hattori, M. and Beppu, T. 2004. Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism. Nucleic Acids Res. 32: 4937–4944. Vance, E. D., Brookes, P. C. and Jenkinson, D. S. 1987. An extraction method for measuring soil microbial biomass C. Soil Biol. Biochem. 19: 703–707. Vivas, A., Moreno, B., del Val, C., Macci, C., Masciandaro, G. and Ben´ıtez, E. 2008. Metabolic and bacterial diversity in soils historically contaminated by heavy metals and hydrocarbons. J. Environ. Monitor. 10: 1287–1296. Weber, S., Stubner, S. and Conrad, R. 2001. Bacterial populations colonizing and degrading rice straw in anoxic paddy soil. Appl. Environ. Microb. 67: 1318–1327. Wertz, S., Degrange, V., Prosser, J. I., Poly, F., Commeaux, C., Guillaumaud, N. and Le Roux, X. 2007. Decline of soil microbial diversity does not influence the resistance and resilience of key soil microbial functional groups following a model disturbance. Environ. Microbiol. 9: 2211–2219. Wilmes, P. and Bond, P. L. 2004. The application of two-dimensional polyacrylamide gel electrophoresis and downstream analyses to a mixed community of prokaryotic microorganisms. Environ. Microbiol. 6: 911–920.